Project Summary Histoplasma capsulatum (Hc) is an understudied fungal pathogen that causes fatal disease in immunocompromised individuals with AIDS. Our long-term research goal is to gain insight into the pathogenic mechanisms used by Hc to kill infected immune cells, ultimately resulting in improved understanding and treatment of Hc infections in HIV-infected individuals. Hc is a soil fungus that is endemic in the Midwestern United States, Central and South America, Africa, and other regions of the world. It is introduced into mammalian hosts by inhalation and is subsequently phagocytosed by macrophages. Unlike most microbes, Hc survives and replicates within the macrophage phagosome. Robust proliferation of Hc within the phagosome is followed by host-cell death, thus allowing live fungal cells to escape from the macrophage and undergo subsequent rounds of phagocytosis and intracellular proliferation. Individuals who lack a cell-mediated immune response are more likely to develop severe disseminated disease, and AIDS patients with Hc infection are subjected to prolonged, sometimes lifelong, anti-fungal therapy. We recently discovered that Hc utilizes the secreted effector Cbp1 to trigger an integrated stress response (ISR) in host macrophages, resulting in host cell death after intracellular fungal replication. The ISR is an intracellular signaling cascade that triggers phosphorylation of the ?-subunit of the translation elongation factor eIF2 as well as induction of the pro-apoptotic transcription factor CHOP in response to a variety of stresses. We have shown that CHOP is required for host sensitivity to Hc infection in the mouse model of infection. These data are now in press at PLoS Pathogens. To our knowledge, these are the first data that implicate the ISR in the host response to fungal pathogens. However, we still have very little understanding of how Cbp1 induces the ISR. Here we propose to build on robust preliminary data to define the mechanism and consequences of Cbp1-dependent ISR induction during Hc infection. We will: (1) investigate the mechanism of how Cbp1 induces the ISR; (2) elucidate if Cbp1 acts alone during Hc infection to induce the ISR and/or host- cell death, or whether other Hc effectors are involved; and (3) determine the mechanism and role of eIF2? phosphorylation, the central signaling event that initiates the ISR, in response to Hc infection. These studies will explore new strategies used by eukaryotic pathogens to control the viability of macrophages, ultimately developing our understanding of general principles deployed by intracellular microbial pathogens to trigger cellular stress and cause disease. Given the susceptibility of AIDS patients to a variety of intracellular pathogens, the establishment of these general principles may be particular relevant in the context of developing therapeutic strategies (such as ISR inhibition) for AIDS-related opportunistic infections with Hc and other like pathogens.